Immunogens comprising capsular saccharide antigens conjugated to carrier proteins are well known in the art. Conjugation converts T-independent antigens into T-dependent antigens, thereby enhancing memory responses and allowing protective immunity to develop, and the prototype conjugate vaccine was for Haemophilus influenzae type b (Hib) [e.g. see chapter 14 of ref. 1]. Since the Hib vaccine, conjugated saccharide vaccines for protecting against Neisseria meningitidis (meningococcus) and against Streptococcus pneumoniae (pneumococcus) have been developed. Other organisms where conjugate vaccines are of interest are Streptococcus agalactiae (group B streptococcus) [2], Pseudomonas aeruginosa [3] and Staphylococcus aureus [4].
Conjugate vaccines for N. meningitidis serogroup C have been approved for human use, and include Menjugate™ [5], Meningitec™ and NeisVac-C™. Mixtures of conjugates from each of serogroups A, C, W135 and Y have been reported [e.g. refs. 6-9], including the Menactra™ product. Other mixtures of conjugated antigens include: (i) meningococcal A/C mixtures [10,11]; (ii) the PrevNar™ product [12] containing seven pneumococcal conjugates; (iii) mixed meningococcal and Hib conjugates [13,14]; and (iv) combined meningococcal, pneumococcal and Hib conjugates [15].
Issues when dealing with conjugate vaccines include stability and batch-to-batch consistency. In Hib vaccines, for instance, catalytic depolymerisation of the saccharide has been reported [16], and conjugates of the serogroup A meningococcus capsule are readily hydrolysed [17]. Instability of conjugates undesirably leads to a reduction in effective dose of immunogenic conjugate over time, variation between batches, and increases levels of uncharacterised breakdown products. References 18 & 19 discuss issues concerning stability testing of Hib conjugate vaccines.
Quantitative glycoconjugate analysis typically involves a first step of saccharide hydrolysis, with analysis then being based on the released monosaccharides. Whereas this analysis is relatively straightforward for single conjugates (e.g. anion-exchange chromatographic methods have been used for analysing hydrolysed conjugates of Hib [20] and serogroup A meningococcus [21]), the situation is more complex in combination vaccines, particularly where different saccharides share monosaccharide units. For example, the capsular saccharides of meningococcal serogroups C, W135 and Y all contain sialic acid, so any method based on measurement of released sialic acid will not be able to distinguish the three serogroups.
It is an object of the invention to provide improvements in quantitative assessment of saccharides in conjugate vaccines for assessing stability and integrity. In particular, it is an object to provide methods that can be used to measure individual conjugates within combined meningococcal conjugate vaccines, and thus to provide improvements in vaccine quality control and consistency.